LDHk is a novel isozyme of lactate dehydrogenase which we have identified in anaerobically shocked rat fibroblasts and in nonrat cells transformed by the Kirsten and Harvey murine sarcoma viruses. It is composed of 56 kilodalton subunits or 35 kilodalton and 22 kilodalton fragments. It is readily isolated from and distinguished from other isozymes in LDH, although we have found it as a contaminant of commercial LDH-5. In biochemical studies of LDHk, we find that oxygen acts by increasing its Km for substrates by approximately 100-fold, while having no effect on its Vmax. This process involves dissociation of subunits. We find that the 5',5'-dipurine nucleoside tetraphosphates Ap4A and Gp4G are exceptionally potent noncompetitive inhibitors of LDHk activity. LDHk acts in a coupled reaction with FAD. Human tumors contain high levels of LDHk activity ranging from 10-\to 500-fold higher than the levels in adjoining nontumor tissue. LDHk has been detected in the serum of over half of all patients examined (greater than 500) but is present in very few noncancer patients. Serum LDHk expression correlates with the presence of metastatic cancer. Studies of LDHk expression in normal tissues have revealed that the retina also expresses high levels of this enzyme. This is seen with mammalian retina but not with retinas of lower species. (A)